Monitoring anthrax toxin receptor dissociation from the protective antigen by NMR
Maheshinie Rajapaksha1, Jack F Eichler, Jan Hajduch
1Department of Chemistry, Wichita State University, Wichita, Kansas 67226, USA.
Abstract:
The binding of the Bacillus anthracis protective antigen (PA) to the host cell receptor is the first step toward the formation of the anthrax toxin, a tripartite set of proteins that include the enzymatic moieties edema factor (EF), and lethal factor (LF). PA is cleaved by a furin-like protease on the cell surface followed by the formation of a donut-shaped heptameric prepore. The prepore undergoes a major structural transition at acidic pH that results in the formation of a membrane spanning pore, an event which is dictated by interactions with the receptor and necessary for entry of EF and LF into the cell. We provide direct evidence using 1-dimensional (13)C-edited (1)H NMR that low pH induces dissociation of the Von-Willebrand factor A domain of the receptor capillary morphogenesis protein 2 (CMG2) from the prepore, but not the monomeric full length PA. Receptor dissociation is also observed using a carbon-13 labeled, 2-fluorohistidine labeled CMG2, consistent with studies showing that protonation of His-121 in CMG2 is not a mechanism for receptor release. Dissociation is likely caused by the structural transition upon formation of a pore from the prepore state rather than protonation of residues at the receptor PA or prepore interface.
Insights
The Bacillus anthracis protective antigen (PA) binding to host receptors initiates anthrax toxin formation. Low pH triggers receptor dissociation from the PA prepore, facilitating toxin entry into cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- Bacillus anthracis protective antigen (PA) initiates anthrax toxin formation by binding host cell receptors.
- Anthrax toxin comprises PA, edema factor (EF), and lethal factor (LF).
- PA undergoes cleavage and heptamerization into a prepore, which transitions to a pore at acidic pH for toxin entry.
Purpose of the Study:
- To investigate the mechanism of receptor dissociation from the PA prepore at low pH.
- To determine if receptor-ligand interactions are maintained during the structural transition to the pore state.
Main Methods:
- 1-dimensional (13)C-edited (1)H NMR spectroscopy.
- Utilized isotopically labeled (carbon-13) and modified (2-fluorohistidine) capillary morphogenesis protein 2 (CMG2) for receptor studies.
Main Results:
- Direct evidence shows low pH induces dissociation of the Von-Willebrand factor A domain of CMG2 from the PA prepore.
- Receptor dissociation was not observed from monomeric full-length PA.
- Protonation of His-121 in CMG2 is not the cause of receptor release, as confirmed by studies with labeled CMG2.
Conclusions:
- Receptor dissociation from the PA prepore is triggered by the structural transition to the pore state at acidic pH.
- This dissociation is crucial for the entry of lethal factor (LF) and edema factor (EF) into host cells.
- The findings clarify the mechanism of anthrax toxin cell entry.
